Glyphosate below no-observed-adverse-effect level exacerbates fatty liver hemorrhagic syndrome in laying hens

Glyphosate (GLY) can disturb gut microbiota homeostasis through inhibiting bacterial shikimate pathway to pose a health challenge to animals and humans. But little information was reported about the influence of GLY on fatty liver hemorrhagic syndrome (FLHS) in laying hens, especially the exposure below no-observed-adverse-effect level. This study aimed to investigate the effects of GLY exposure on the progression of FLHS using a laying hen FLHS model induced by high-energy low-protein diet (HELPD). Ninety healthy Hyline brown laying hens were randomly allocated into 3 groups and subjected to the following feed for 6 weeks: Control group (fed with a basal diet), FLHS group (fed with HELPD), and FLHS + GLY group [fed with HELPD containing GLY (equal to 47 mg/kg body weight/d)]. Fresh feces were collected for microbiome and metabolome analysis, as well as fecal microbiota transplantation (FMT) experiments. The liver was collected for differentially expressed gene analysis and the colon for gut permeability determination. The results revealed that GLY exposure aggravated liver damage and lipid deposition by reshaping gut microbiota and increasing gut permeability in FLHS hens. Specifically, GLY exposure significantly elevated the relative abundance of Escherichia-Shigella, Bacteroides, Erysipelothrix, Desulfobvibrio, and Eggerthellain, while markedly decreased the relative abundance of Lactobacillus and Bifidobacterium, thus instigating the decline of certain tryptophan metabolites, including 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, kynurenic acid, 4-(2-aminophenyl)-2,4-dioxobutanoic acid, tryptamine, and indole-3-carboxylic acid in the gut, which inhibited aryl hydrocarbon receptor pathway to impair gut integrity. The impaired gut barrier led to the increase of serum LPS level, triggering inflammation and oxidative stress in the liver via activating TLR4/MyD88/NF-κB pathway and inhibiting Nrf2/Keap1 pathway. Furthermore, FMT experiments substantiated that GLY exposure aggravated FLHS in a gut microbiota-dependent manner. Collectively, these findings demonstrate that GLY exposure aggravates the progression of FLHS in laying hens through modulating the gut-liver axis. This study provides theoretical basis for assessing adverse effects of GLY exposure below no-observed-adverse-effect level on animal welfare.